The Danger model has two parts. The first part, which is the subject of this report, deals with the first question the immune system must answer when faced with a potential threat: namely shall I respond or not?. The second question (what kind of response should I make) is the subject of the other report from our lab. The Danger model suggests that the immune system is called into action when a damaged or stressed tissue sends alarm signals that activate antigen presenting cells. This view has had a lot of support and, with the discovery that mitochondria can act as alarm signals, brings together the Danger model and the other current major theory of immunological control, the PRR model of Charlie Janeway. Janeway suggested that the immune system is called into action by the recognition of evolutionarily ancient conserved molecules on bacteria. Mitochondria fit that bill perfectly, as they are both internal cell components, and are ancient bacteria. We have been working on the signals that activate the immune system in several ways. PROJECT 1) IMMUNE ACTIVATION LEADING TO CERVICAL CANCER More than 97% of women infected with HPV clear the virus and remain protected for life. Of those that do not clear the virus, a very small percentage, many years after infection, contract cervical cancer. It has been shown that the cancer cells contain Human Papillomavirus (HPV) that has integrated into the genome, and lost most of the virus copies that normally exist in the cytoplasm of infected cells. In the process of integration, the virus loses a gene called E2 (early gene #2) and this loss is instrumental in the generation of the cancer cells. The reason is that E2 is a suppressor of the genes E6 and E7, which themselves are suppressors of two anti-tumor genes that are critical for maintaining cellular health (p53 and retinoblastoma). In the absence of E2, the E6 and E7 genes are liberated, and this leads to chromosomal instability and, eventually, cancer. However, most HPV infected cells carry a number of viral copies in the cytoplasm, and the E2 gene products from these cytoplasmic viruses are sufficient to maintain suppression of E6 and E7. It is only when the cytoplasmic copies are lost that the integrated E6 and E7 become active. It has been suggested that the loss of the cytoplasmic copies of the virus is the result of an immune response, yet there has been no explanation why, after 15 years or so, a woman that did not make a good enough immune response to clear the virus suddenly generates a response that clears it from a small sample of cells, while leaving it intact in many others. Taking on on the challenge of deciphering the events that initiate the immune response that leads to cytoplasmic virus loss, we analyzed data from over 500 patients (ranging from early pre-cancerous lesions to advanced tumors) using a combination of chromosomal aberration studies and new methods of assessing gene networks from mRNA expression. We found that cervical tumors almost invariably contained changes in two gene networks one involved in the anti-viral response both intracellular (such as IFN dependent responses) and extracellular (such as recruitment of activated immune system cells), and the other regulating the cell cycle. Further, the random chromosomal aberrations found in the tumors almost invariably included amplifications of the regions housing key regulators of these networks. Advanced tumors were highly enriched for chromosomal aberrations causing the amplification of both types of genes. Altogether the data suggest that, over the years, HPV infected cells accumulate random chromosomal aberrations. The rare cell in which the aberrations induce amplification of the antiviral response will lose its cytoplasmic virus, freeing E6 and E7 to suppress p53 and retinoblastoma. If the cell also houses an aberration that causes deregulation of the cell cycle, it has a very high probability of becoming a tumor. PROJECT 2) CD4 T CELLS CLEAR TUMORS a) CELLULAR PARTNERSHIPS IN TUMOR CLEARANCE: Having found that CD4 T cells can be better at clearing tumors than CD8 cells (gainst six out of six tumors, from five different tissues, CD4 effectors were more potent than CD8s), we began stuyding the mechanisms used by the CD4 T cells. We found that the CD4 T cells partner with NK cells and with a macrophage population in tumors that has characteristics of both macrophage and dendritic cell. We are now studying these partnerships to determine which cells do what. Thus far, it seems that IFN-gamma production by the CD4 T cells is involved, and that tumor infiltrating macrophages can be educated by CD4 T cells to change from tumor-nurturing to tumorocytic. b) COLLABORATION BETWEEN TUMORICIDAL LYMPHOCYTES AND TUMOR-RESIDENT MACROPHAGES. We found that activated CD4 T cells change the phenotype of tumor-resident macrophages from tumor-promoting to tumoricidal. macrophages isolated from tumors that have been treated with CD4 T cells have an M1 pattern of cytokine secretion, as compared to macrophages from untreated tumors, which secrete M2-type cyotkines.